Electrically conductive carbon‐based (bio)‐nanomaterials for cardiac tissue engineering

Abstract A proper self‐regenerating capability is lacking in human cardiac tissue which along with the alarming rate of deaths associated with cardiovascular disorders makes tissue engineering critical. Novel approaches are now being investigated in order to speedily overcome the challenges in this path. Tissue engineering has been revolutionized by the advent of nanomaterials, and later by the application of carbon‐based nanomaterials because of their exceptional variable functionality, conductivity, and mechanical properties. Electrically conductive biomaterials used as cell bearers provide the tissue with an appropriate microenvironment for the specific seeded cells as substrates for the sake of protecting cells in biological media against attacking mechanisms. Nevertheless, their advantages and shortcoming in view of cellular behavior, toxicity, and targeted delivery depend on the tissue in which they are implanted or being used as a scaffold. This review seeks to address, summarize, classify, conceptualize, and discuss the use of carbon‐based nanoparticles in cardiac tissue engineering emphasizing their conductivity. We considered electrical conductivity as a key affecting the regeneration of cells. Correspondingly, we reviewed conductive polymers used in tissue engineering and specifically in cardiac repair as key biomaterials with high efficiency. We comprehensively classified and discussed the advantages of using conductive biomaterials in cardiac tissue engineering. An overall review of the open literature on electroactive substrates including carbon‐based biomaterials over the last decade was provided, tabulated, and thoroughly discussed. The most commonly used conductive substrates comprising graphene, graphene oxide, carbon nanotubes, and carbon nanofibers in cardiac repair were studied.

conceptualize, and discuss the use of carbon-based nanoparticles in cardiac tissue engineering emphasizing their conductivity. We considered electrical conductivity as a key affecting the regeneration of cells. Correspondingly, we reviewed conductive polymers used in tissue engineering and specifically in cardiac repair as key biomaterials with high efficiency. We comprehensively classified and discussed the advantages of using conductive biomaterials in cardiac tissue engineering. An overall review of the open literature on electroactive substrates including carbon-based biomaterials over the last decade was provided, tabulated, and thoroughly discussed.
The most commonly used conductive substrates comprising graphene, graphene oxide, carbon nanotubes, and carbon nanofibers in cardiac repair were studied.  [3][4][5][6][7] Other than organ transplant and pharmaceutical approaches, surgical reconstruction procedures effectively aim for tissue repair. It mainly focuses on controlling inflammation, [8][9][10] reducing scar formation, 11,12 and identifying cures for fibrotic diseases and chronic wounds. 13,14 Total artificial substitutes (such as artificial joints) and nonliving processed tissues (such as heart valves) are the replacing strategies that are pleasingly efficient. In addition, harvested flaps (including autografts or allografts) are conventional strategies associated with reconstruction perspectives. 15,16 However, harvesting autografts is usually accompanied by challenges of formidable donor site morbidity. Besides, it requires multiple separate operations, which is preferably avoided. On the other hand, in the course of applying grafts, precisely transplanting vasculatures to the target site demands highly accurate and advanced equipment. High risks of infection or disease in case of allotransplantation can also arise. 17 Hence, surgical reconstruction procedures as well, may not be the method of choice when it comes to urgent and critical situations.
Tissue engineering is a promising technique aiming at tissue reconstruction through regeneration. The three main approaches are cell transplantation, matrix-guided regeneration, and simultaneous utilization of both cells within matrices. Nowadays, tissue engineering employs an optimal combination of cells, substrates, and bioactive molecules to alleviate lost tissues. 15,18,19 Scaffolds are assumed to act as a matrix aiming to satisfy several demands, primarily providing the initial cell support. 20 They are supposed to adhere to cells via ligands and chemical groups/compounds of atoms. [21][22][23] Thus, hydrophilic materials, 24,25 porous structures, [26][27][28] and large specific surface areas 29 efficiently facilitate cell adhesion.
New blood vessel formation, including vasculogenesis and angiogenesis, is also a critical challenge that promotes cell survival and enables the operation of larger tissues. Previously mentioned demands and pores interconnectivity, which promote mass transport including oxygen and nutrient transfer, and the integration of the implant to the adjacent area are likely to be associated with interactions with the microenvironment. [30][31][32] Moreover, scaffolds and matrices are supposed to function as mechanical support during tissue formation, whether in vitro or in vivo. Therefore, an appropriate elasticity and stability in the case of either soft or hard tissue are essential. Optimization between the density, porosity, and mechanical properties of the scaffold is of great importance as a consequence. Apart from this, while tissue growth and adhesion occur, concurrent degradation of the scaffold occurs with extracellular matrix proteins replacing it. 33 Nontoxicity and ease of absorbance or excretion of degradation products are other vital factors.
Applied materials acting as cell bearers are extremely fundamental because of different reasons. As indicated, they are employed as supporting substrates acting as an appropriate microenvironment for the specific seeded cells. Additionally, a substrate protects cells from being recognized by the immune system 34 and neutrophils attack 34,35 of the patient's body. Depending on the native tissue, the composition, elasticity, and microstructure of the extracellular matrix (ECM) differ explicitly from tissue to tissue and even in different periods of one specific tissue. Consequently, it has been demonstrated that cultured cells on various substrates with differing features show various responses. 36 Thus, to eventually accomplish the expected cellular behavior, multiple parameters should be regarded. 20 Considering every stated parameter, deciding on an appropriate substrate in tissue engineering is of tremendous importance. Acellular tissue matrices, biocompatible natural or synthetic polymers, ceramics and their composition, and recently graphene-based materials are considered suitable choices for the substrates. Material selection is based on the application, cell-scaffold interactions, appropriate mechanical and electrical properties, required time of the scaffold performance before degradation, and the feasible fabrication methods. [37][38][39][40][41] This review aims to provide a survey on cardiac tissue engineering and the significance of conductivity at the same time. First, electrical conductivity was defined and different aspects of such characteristics in nanomedicine were discussed. Then, conductive polymers used in tissue engineering, particularly in cardiac repair, were comprehensively classified and their advantages in cardiac tissue engineering were highlighted. As the main objective of this work, an overall review of studies on electroactive substrates comprising carbon-based materials within the past few years was reported, tabulated, and discussed.
In this regard, the most frequently used carbon-based substrates including graphene, graphene oxide, carbon nanotubes, and carbon nanofibers in cardiac repair were studied.

| CARDIAC TISSUE REGENERATION
Different biomaterials and their combinations are currently under investigation for tissue engineering. 42 Decellularized tissues have been widely used as either cell seeding or cell-free substrates. 27 Extracellular matrix-derived materials are beneficial since they provide a native microenvironment for the specific cells to survive, proliferate, and differentiate. 43 Native ECM mixture supplies specific molecules and proper structure, promoting cell phenotype and maintaining tissuespecific ECM construction. However, ECM variations originating from differing donors, immunologic and inflammatory response of the recipient, possible rejection of the implant, and regulatory issues, are the topics of limitation. 19 Naturally-occurring polymers, on the other hand, can be extracted from living organisms. Collagen, cellulose, alginate, silk fibroin, and chitosan 44 are among the favored natural polymers typically used in this field. These polymers are beneficial due to their biological inherent mimicking of natural ECM structure. 45 In return, lack of proper mechanical strength and hardly controllable degradation rate is a considerable drawback of natural materials. Apart from that, potential contaminants may be presented within the structure of natural polymers (S. J. Lee et al., 2018), such as heavy metals, formaldehyde, polyphenolic compounds, and bacteria, and this brings about the possibility of pathogenic behavior for such an eventually. 46 Natural polymers and acellular matrices are beneficial in the aspect of biological recognition; while synthetic biomaterials provide the potential for more flexible and controllable properties such as mechanical characteristics and degradation rate. 47 Mostly applied synthetic polymers used for tissue engineering include poly(ethylene glycol) (PEG), poly(ε-caprolactone) (PCL), poly(lactide) acid (PLA), poly(lactic-co-glycolic acid) (PLGA), and polyurethane (PU). They are advantageous in the aspect of flexible physical and chemical properties. However, potential cytotoxicity due to lack of biological inherency is a disadvantage likely to promote cell reaction. 48 Due to their substantial potential for osteoconductivity, ceramics are the material of choice for repairing and regenerating musculoskeletal and periodontal disorders. This is due to ceramics' fine biological and mechanical properties such as biocompatibility, hardness, and corrosion resistivity. 49 Major hurdles in employing ceramics as substrates are attributed to their brittleness and high Young's modulus, making them difficult to process. 50 Bioceramics are generally serving as in three categories. Bioinert ceramics such as alumina and zirconia are used when no interaction between the implant and the environs is preferred and is ascribed to the relatively high corrosion and wear resistance. Bioactive ceramics, on the other hand, gradually join their surroundings through osteogenesis. Bioactive glasses and glass ceramics are grouped into this category. Lastly, biodegradable ceramics, resorbed within the body over time, such as calcium phosphate-based ceramics, are known as bioresorbable ceramics. 51 Cardiovascular diseases (CVDs) are undoubtedly the primary cause of death globally, in recent years. 52 Nearly 18 million deaths in 2016 were attributed to CVDs which is around 31% of all deaths.
Except for the significant health threat, CVDs are a significant economic burden. According to the statistics, CVDs consume 14% of the USA health care cost annually, so 189.7 billion $ has been directly spent from 2012 to 2013 on the CVDs for direct expenditure with respect to 316.1 billion $ spent indirectly. It is anticipated that an unbelievable budget of about 918 billion $ will be demanded for the CVDs by 2030. 53 Many CVDs are identified nowadays, such as stroke, rhythm disorders, heart failure, cognitional heart disease, and atherosclerosis.
Congenital heart defects are among the most common congenital disabilities. 54 More than 24% of infants dying due to a congenital disability suffer from congenital cardiovascular defects. Aside from that, coronary artery disease (CAD), also known as ischemic heart disease, is the most common class of CVDs 55 associated with a partial blockage in major coronary arteries due to atherosclerosis.
In early diagnosis, coronary artery disease is finely treated with the percutaneous coronary intervention technique, also known as angioplasty. It is a nonsurgical procedure in which a catheter is inserted into blood vessels (usually the femoral artery in the thigh) and guided up toward the heart into the considered coronary artery.
A balloon catheter is then pushed into the area and inflated to pull over the blockage and widen the vessel. Finally, a stent is placed to ensure the vessel remains extended since the balloon is ejected. 56 Coronary artery bypass grafts may be required if the situation is more severe. An occluded coronary artery is bypassed utilizing an isolated artery or vein graft through a surgical procedure. The graft, usually harvested from the patient's leg or chest, is transplanted into the area with inadequate blood supply to provide a new pathway for blood flow. The heart may need to stop beating during the procedure and be timely replaced by a heart-lung machine. 56 As the blockage gradually intensifies, a severe occlusion typically forms if not diagnosed or treated properly, causing a significant heart attack due to improper expansion and contraction of the myocardium.
After a heart attack, fibrotic scar tissue will be generated because of the limited capacity of myocardial tissue in inherent regeneration, which such incapability ends in left ventricular dysfunction and cardiac arrhythmias. 57 Myocardial infarction (MI) occurs due to progressive, disrupted coronary circulation and unstable angina-deficiency of blood flow within the heart muscle. It is followed by a significant loss of cells in the dedicated area in response to oxygen demand and supply imbalance. 58 The amount of involved area depends on the size of the coronary artery, the occlusion severity and duration, and the level of demanded oxygen by the involved myocardium.
Local cell death occurs due to the inadequacy of blood supply and oxygen shortage, ischemia in shorts. This occurs through the entire or part of the myocardium thickness within the involved area. The body's inflammatory response immediately begins, 13 aiming for tissue repair leading to prompt healing. Cardiac muscle is explicitly made up of different cell types assorted as myocytes and non-myocytes, including cardiomyocytes (CMs), fibroblasts, endothelial cells, and peri-vascular cells. Although 70%-85% of the volume of cardiac muscle is occupied by CMs, these cells represent only 30% of the whole cardiac cell population. 59 Hence, the inability of cardiac muscles to self-regenerate is likely attributed to the CMs limited capability to proliferate in practice. 60 In other words, an abundant potential for renewal lacks within cardiac contractile cells. 61 Since the constant function of the myocardium is crucial, 41,52 healing rapidly recovers the deficiency of lost cells to compensate for the insufficiency. Thus, necrotic tissue formation and collagen deposition overtake regeneration, taking part in the healing process. 62 Shortly after MI, several inflammatory responses are followed. In brief, oxidative stress, represented as enhanced generation of oxygen radicals or reactive oxygen species (ROS), is rapidly established. Meanwhile, inflammatory cytokines such as TNF-α, IL-1β, and IL-6 are produced ( Figure 1). Subsequently, cardio depressive reactions take place. Furthermore, the activation of matrix metalloproteinase (MMP) enables ECM remodeling. After that, collagenous tissue formation and fibrosis take place. Myocardium remodeling and LV dilation are also long-term outcomes. 63 As a result of cell necrosis, the body's inflammatory response is accompanied by ECM degradation to elucidate the indicated phenomena. This allows neutrophils and macrophages migration to the infarcted area. Phagocytosis of necrotic cells takes place as a consequence. Later, proliferating fibroblasts and endothelial cells establishing the granulation tissue replace necrotic cells. 64,65 Healing continues with the transformation of granulated tissue into scar tissue within a month or so. Rearrangement of cells and ECM to compensate for the injury causes a disturbance in the integrated electrophysiological performance of cardiac muscle. Given the aforementioned subsequent circumstances, uncoupled, dense, collagen-rich scar tissue with independent mechanical and electrical properties than natural myocardium appears. Changes in ion channels and intercellular gap junctions are followed. Accordingly, lost integrity through the electrical activity of heart muscle causes a cardiac rhythm disturbance. 13,35 Taken together, these impairments lead to the insufficient capacity of the heart muscle to pump enough blood throughout the whole body. Due to the previously mentioned circumstances, mechanical stress brings about several permanent outcomes, including ventricle enlargement, heart wall thinning, geometry change, and LV dilation.
LV chamber gradually encounters a minor conversion in its overall shape from ellipsoidal to spherical. This possibly leads to mitral regurgitation. Changes in the cavity diameter, mass, and geometry of the heart muscle bring about adverse impacts and deficiencies in cardiac The molecular events occurring during myocardial infarction include ROS overgeneration that can lead to oxidative stress, and enhanced levels of cytokines such as TNF-α, IL-6, and IL-1β for mediating inflammation. Furthermore, MMPs can mediate ECM remodeling during myocardial infarction.
performance. If so, chronic heart failure is likely to be inevitable then. 63,66 Drug treatments for patients either already suffering from or likely to face such difficulties within the near future, including betablockers, ACE inhibitors, and angiotensin receptor blockers, are the most common treatments. However, they are not efficient enough, counted as inhibitors of LV dilation. 63,[67][68][69][70] Total heart transplantation, on the other hand, is by far a satisfying approach though insufficient donors, heightened risks of open-heart procedure, probability of organ rejection, complex postoperative cares, and precautions of immunosuppression regimens are still considerable challenges making this approach extremely complicated. 34,[71][72][73][74] Moreover, such surgical operations carry significant risk to older people, which suffer from CVDs more often than younger patients do. Therefore, alternative regeneration routes have emerged to repair heart function appropriately. New emerging methods should be noninvasive (eliminating heart surgery), affordable, efficient, and appropriate for mimicking cardiac tissue.
As a solution to this unmet demand, myocardial tissue engineering with the aim of cardiac regeneration raises the chance for a total replacement of the injured tissue and a perfect reliable approach. 75 Myocardial tissue engineering approaches, as reported by Chen et al., 76 mainly include cell-based therapy, scaffold-free cell-sheet implantation, heart patch implantation, and 3D tissue engineering construction. Cellular-based therapy employs suspended progenitor or stem cells in saline or culture medium injected into the infarcted area. 77,78 However, cell survival is disappointing on this occasion regarding poor cell adhesion within the infarcted area. This is mainly due to the raised concentration of ROS inhibiting cell adhesion following MI. 79 Cell-containing or cell-free matrices and cardiac patches are approaches in which the task is to mechanically support the infarcted myocardium to prevent dilation and induce regeneration. This has been shown to effectively slow down the remodeling process and scar formation. 80 Different strategies to regenerate the cardiac are shown in Figure 2. 81 CMs are the most suitable cells to be delivered in cell-based therapy. 35 However, the major hurdles are poor cell integration with native tissue and thus disappointing cell retention rate. 71 Moreover, the shortage of a reliable cardiac-specific cell source and ethical issues attributed to fetal or neonatal CMs are also principal hindering issues.
Stem and precursor cells favor sources and differentiated cells, yet there are various particular challenges to overcome. Accurate control of cell differentiation or conversion, teratocarcinogenicity, and concerns associated with allogenic sources are formidable limitations. 35,82 F I G U R E 2 Cardiac tissue engineering strategies. Cells, scaffolds, and signaling molecules can be introduced alone or in combination at the injury site. Scaffolds provide biophysical, topographical, and biochemical microenvironments to the transplanted and host cells. The mechanical stiffness of biomaterials can guide proper stem cell differentiation. Stretch is a typical function of the cardiovascular system and has been shown to guide the differentiation of stem cells toward cardiomyocytes (CMs) or smooth muscle cells. Nanotopography of the biomaterial can affect stem cell phenotype, cellular alignment, and electrophysical properties 81 Efficient recruitment of appropriate cell types and selecting a proper substrate to enhance cell retention and integration, as a result, is highly pivotal.
In brief, cell therapy and tissue engineering are seeking induction of regeneration. Accordingly, different demands should be met in order to improve cardiac performance efficiently. Selection of appropriate matrix, composition, microstructure, chemical and mechanical properties, and cell-matrix interaction makes huge differences. 20 Cell type and the origin, potency, surface markers, combination, population, dispersion, cell-cell contacts, cell signaling, and gene and protein expression are also required to be determined. 83 Cell cultivation in vitro before transplantation has been demonstrated to be promising compared to direct delivery of precursor cells. Improved cell retention, survival, and integrity are guided by precultivation. 82,[84][85][86][87] Designing a proper structure for cardiac regeneration requires profound knowledge about the cardiac structure, function, and interaction with biomaterials. 88 The heart is composed of four chambers dividing into ventricles and atria encased in the pericardium. Deoxygenated blood is collected in the right atrium and then is passed through to the right ventricle. Once oxygenation of the blood is completed by contracting and pumping through the lung, it will be collected in the left atrium and takes the way toward the right ventricle.
The wall of the heart ( Figure 3) includes three strata: the endocardium, epicardium, and myocardium. The interlayer is the endocardium that lies between ventricular and atrial. The myocardium is the middle layer composed of the muscular component of the heart wall. It is dense lamellar, vascularized, oriented, interwoven within collagen, and conductive. The outermost layer is the epicardium. 90,91 A heart pacemaker is a sinoatrial (SA) node, a small bunch of node cells with a high intrinsic depolarization rate. It lies between the myocardium and the epicardium, juxtaposing the right atrium. Such a node generates the electrical current and sinus rhythm, which contracts the heart and establishes the normal cardiac rhythm, the most mysterious part of heart mechanics. SA, by the aid of internodal pathways (IP), spreads impulses throughout the atria. Three bands of IP, including anterior, middle, and posterior, are conducted in juxtaposing nodes in 50 ms time intervals in which myocardium contractile cells can deliver an impulse to the atrioventricular node using a cell-by-cell pathway. 19,43,92,93 Moreover, impulse straightly is conducted from the right atrium to the left atrium using Bachmann's bundle. By reaching the impulse to the atrioventricular septum, the spreading of the impulse to the myocardial cells is inhibited by the connective tissue of the cardiac skeleton. Na + , K + , and Ca 2+ play essential roles in generating the action potential (electrical impulse). Available sodium channels on conductive cells result in gentle sodium ion flux, which causes to ascend the membrane potential from À60 mV to À40 mV. Such movement of ions causes automatic depolarization. After that, the Ca 2+ gate opens, and ions enter the cell and depolarize to reach +5 mV. Then, repolarization happens by opening the K + channels and closing the Ca 2+ channels whose membrane potential reaches À60 mV. 94,95 Wide ranges of biomaterials have been utilized so far to mimic the physiochemical properties of cardiac tissue. Table 1 presents the literature reports on the scaffolds applied in cardiac tissue engineering. It can be seen that all of them showed significant disadvantages despite the seemingly very promising properties. One of the flaws was the lack of conductivity, so these materials cannot substitute myocardium. Therefore, conductive biomaterials have received substantial importance thanks to their inherent feature that recapitulates the cardiac tissue characteristics. 105,106 CMs' functionality is improved using conductive substrates (with and without electrical stimulation) because of the cardiac synchronizing. 107,108 In this regard, the architected scaffold should recapitulate the 3D anisotropic structure of the heart to provide a proper milieu for cellular activity. Bundling the undulated fiber of perimysial collagen inside the honeycomb-like structure forms an endomysial collagen layer that surrounds the cardiac muscle fibers. 109,110 Such structure endows the anisotropic features with mechanical and electrical characteristics.
Various classes of the 3D structure have been designed over the years to mimic the heart function by taking credit for maximum  89,114,115 Concluding, electrical conductivity is a crucial factor in scaffolds fabricated for cardiac tissue engineering. Due to the specific electrical properties of cardiac tissue, in which contractility is the result, the electrical conductivity of the construct, signal propagation, and synchronous contraction capability also merit consideration ( Figure 5).
Electroactivity is defined in further detail within the next part.

| ELECTRICAL CONDUCTIVITY
In an attempt to tissue repair and regeneration, engineered constructs mimic the original niche through their specific features. 120,121 This requires an appropriate combination of the designed construct with particular mechanical, physiological, and electrical properties, similar to the native tissue. Since any communication, including scaffold and cell receptors' interaction, cell-cell signaling, and intracellular activities, is disposed to be engaged in a compelling performance, electroactive substrates in which cells are seeded for tissue engineering efficiently promote cell behaviors and regeneration. [122][123][124][125][126] A specific voltage across cell membranes specifies the resting potential and ion exchange and varies depending on cell type and tissue. 127,128 Hence, regulation of the ion exchange highly impacts cell behaviors, including cell attachment, cell proliferation, protein expression within cells, and cell maturation. Less resting potential through cell membranes induces more proliferative capacity, as observed in cancer and stem cells. 127 Thus, an appropriate conductivity of the designed construct in tissue engineering regulates ion transfer resulting in enhanced cell proliferation. 127 Figure 6 shows that conductive biomaterials, according to adaptability, can be designed in order to target tissue to improve regeneration. Substrate conductivity, which can be accustomed by synthesis assay, can affect drug release design, and cell behavior. 129 Bone tissue regeneration, for instance, is electricity attributing.
Applied mechanical forces to bones induce an electrical field owing to the piezoelectricity characteristic. 130,131 Apparently, electrical stimulations induce cell proliferation and bone healing. [132][133][134] Promoted biomineralization, accelerated formation of tri-calcium phosphate, improved cell proliferation, and osteogenic differentiation has been observed in conductive bone matrices in contrast to nonconductive ones. 135,136 T A B L E 1 Scaffolds utilized in cardiac tissue engineering, their fabrication method, properties, and challenging disadvantages Conductivity is also recommended regarding neural and cardiac tissue regeneration. 125 Data transfer is conducted by an action potential within neural networks, requiring a conductive substrate. 137,138 Likewise, upregulated expression of neural progenitor markers, enhanced cell differentiation toward neurons, and promoted neural induction within conducted substrates have been demonstrated. [139][140][141][142][143] Other than neural tissue, muscles' contraction is also followed by an electrical signal propagating throughout the tissue. In cardiac tissue repair, the conductive substrate is in charge of electromechanical and electrochemical transmittance leading to electrical stimulation of cells. Synchronized contractions are attainable as long as the propagation of electrical impulses is achieved. 144,145 It has also been demonstrated that conductivity in cardiac tissue engineering modulates cellular function and enhances cardiac gene expression. 60,146,147 In contrast to skeletal muscles in which contractions are neurogenic, smooth and cardiac muscle contractions are myogenic, initiating from the heart itself, along with a rhythmic and autonomous behavior.
Contractions within cardiac muscle generally arise from impulses gen- The conducive platform's properties are adjustable with various tissues. The plot on the left-hand side advises on the selection of biomaterials for a target tissue considering their conductivity and mechanical properties, while the right-hand one provides the investigator with a quick view of the microstructure-property-performance relationship when one takes the first step in the selection of conductive biomaterials for tissue engineering and regenerative medicine uses. 129 through the walls of the ventricles. This order is likely because Purkinje fibers originate from the inner ventricular septum and extend to the papillary muscles toward the ventricles' walls.
As indicated, cardiac muscle conductivity is mainly attributed to the conducting Purkinje fibers, 129  Interruptions through intercalated discs thus uncoupled CMs and disrupted contractions, as already defined previously, are one of the main complications of MI. 153 Efficient treatments to the ischemic myocardium approaching tissue regeneration have to meet different demands, among which electrical conduction has been widely studied.
Due to the heart muscle anatomical structure, an anisotropic, discontinuous electrical conduction 154 is reported to match the amount of 1.6 Â 10 À3 S cm À1 along and 5 Â 10 À5 S cm À1 across the myocardium. 138,[155][156][157][158] Studies have furthermore confirmed the influence of electrical conduction and stimulations on the regenerative behavior of body tissues. Cell division, tissue growth, and wound healing, as evidenced by studies, are observed to be obviously affected. 159 Electrical conductivity comes after moving ions, carrying charge in one or more directions within the substance. It is provided by the flow of negatively charged electrons and positively charged holes. Seeking tissue regeneration, conductive materials, or incorporated electroactive particles and other materials are employed to promote electroactivity. Conductive polymers, metallic nanoparticles, and carbon-based materials are currently the standard choices in this field. Depending on the material selected and the application, optimization is always necessary to reach good electrical, mechanical, and biological properties. 160 Some polymers require a doping process in order to be modified as conductive materials. 144 Predominant conductive polymers used in tissue engineering include polypyrrole (PPy), 161,162 polyaniline (PAni), [163][164][165] polythiophene (PTH), and its derivatives. [166][167][168][169] Apart from their proper conductivity, the use of conductive polymers bears disparate advantages, including producibility, processability, surface modification potency, relatively low cost, and suitable biocompatibility. Meanwhile, comparatively poor solubility and challenging biodegradability demand further consideration. 160,162 In cardiac tissue engineering specifically, a close elasticity resemblance to the native myocardium is essential owing to the frequent contractions of the heart. Polymers are likely to exhibit an undesired rigidity which makes their use limited in this field. 155,158 Other than conductive polymers, prevalent metallic nanoparticles widely used in biomedicine are gold, [170][171][172][173][174] copper, 175,176 and silver [177][178][179][180] nanostructures. High electrical conductivity, high surfaceto-volume ratio, ease of synthesis, and magnetic and antibacterial properties have disposed of metallic nanoparticles to be engaged in the area of tissue engineering and regenerative medicine. Contrastively in a long-term spectrum, cytotoxicity is the foremost hurdle making biocompatibility of these materials a severe challenge. 180,181 In addition to the materials mentioned above, carbon-based materials also have the magnificent potential to result in electroactivity. These include graphite, 182,183 graphene, 184-186 graphene oxide, 187,188 reduced graphene oxide, [189][190][191] carbon nanofibers, 60,[192][193][194] carbon nanotubes, 156,195,196 fullerene, 197,198 carbon quantum dots, [199][200][201] and nanodiamonds. [202][203][204] Particular mechanical, electrical, thermal, and optical properties bring about the opportunity for carbon-based nanomaterials to be involved in the field of tissue repair. 205,206 Conductive carbon-based polymers, as well as carbonbased nanomaterials and their application in developing electro-active cardiac tissues, are discussed in detail in the following parts.

| CONDUCTIVE POLYMERS
The simplest method to provide the electroactivity of scaffolds and other materials used in cardiac tissue engineering, which should be characterized by an efficient cellular response, is the application of conductive polymers. 207,208 In general, conductive polymers mainly include polypyrrole. 209 polythiophene, and the most well-known member of this family, polyaniline (PANI). 210 These polymers have shown promising features for the regeneration of electrically responsive tissues. 90 The conductivity mechanism of inherently conductive polymers is ascribed to the sequential sp 2 hybridized carbon existing in their structure. 211 Combination of Pz orbital with residual valence electron results in delocalized orbitals allowing electrons to move freely during the doping process. Oxidative and reductive doping yields p-type and n-type conductive/semi-conductive materials. Typically, the conductivity of such polymers can be tuned from 10 À6 to 10 2 S/cm. The primary cell functions, such as attachment, proliferation, migration, and differentiation, could be modulated through electrical stimulation. 212 Figure 7 displays an overall view of conductive polymers' usage in nanomedicine. Some successful attempts can be found in well-established reports devoted to neural, 213 bone, 214 skin, 215 and more specifically, cardiac tissues. 216 However, there have been confusing reports on the biocompatibility function of conductive polymers and the cytotoxic characteristics of such materials. 147 Polypyrrole is one of the favorable conductive polymers widely utilized as a biomaterial. 49  wound dressing. 165,223 In this sense, research directed at the usage of PANI in cardiac regenerative nanomedicine was stressed in this part.     Degradation studies on prepared samples in PBS medium at 37 C showed more weight loss in composites than pure PGS over 30 days. The rigidity of the EM AP made it difficult for the GA chains to freely coil by coiling around the template of the EMAP aggregate. It was found that with increasing the content of AP graft, the degradation rate was decreased. GA lost about 80% of its weight at 28 days; however, AP-g-GA polymers did not experience weight loss of more than 65%. The authors stated that the hydrophobicity and steric hindrance of AP-g-GA increased by introducing AP to GA, but still, it was considered biodegradable. With increasing the content of AP in AP-g-GA copolymer, the cell viability diminished slightly. However, pure EMAP exhibited low cell viability compared to that of GA and AP-g-GA. The improvement in cytocompatibility of the AP-g-GA was ascribed to the biocompatibility of the gelatin. The degradation products of AP-g-GA also showed no cytotoxicity with a slight decrease when the concentration was 50 mg ml À1 . It was stated that the introduction of more AP in the structure of copolymer could increase the charge and the toxicity at the same time. While electroactivity could accelerate cell proliferation, but toxicity has a negative influence. Therefore, high percentages of AP may not be suitable for polymers used as biomaterials. The authors stated that beyond the electroactivity, the introduction of AP changed the irregular structure of the scaffolds to a very regular one, which may be used as a template for the normal differentiation of neuronal or cardiovascular cells (Figure 9).
Researchers have also successfully synthesized novel biodegradable electroactive polyurethanes containing aniline pentamer (AP-PU) for cardiac tissue engineering uses. 163 The AP-PU was blended with PCL at an equal weight ratio to tune the physicochemical properties and biocompatibility. The electrical conductivity of the prepared samples was recorded in the semiconductor range (~10 À5 S/cm). It has been proved that the conductivity of about 10 À6 S/cm is sufficient to conduct micro-current for stimulating neuronal cell proliferation and differentiation since the human body has a lower micro-current intensity. 252 However, the semiconductor range of conductivity was still enough. 161 MTT assays using L929 mouse fibroblast and HUVECs showed that the prepared blend (PB) displayed more cytocompatibility than AP-PU due to the introduction of a biocompatible PCL moiety. The in vitro cell culture also confirmed that PB was as supportive as the tissue culture plate. However, AP-PU with the higher AP concentration showed less compatibility than PB containing lower amounts of AP. Therefore, optimization of AP concentration is imperative for acquiring specimens with the most negligible cytotoxicity. The evaluation of the antioxidant activity of conducting polymers and nanomaterials needs to be taken into account when these materials are considered for biomedical applications. 253

| CARBON-BASED NANOMATERIALS IN CARDIAC REPAIR
Although using conductive carbon-based nanomaterials seems a simple way to make the scaffolds conductive, problems associated with size-dependent, 254 shape-dependent, 255 environment-dependent, 256 cell-dependent, 257 and/or performance-dependent 258   give rise to the photoluminescence radiation feature in CDs, which in addition to electrical conductivity, low cytotoxicity, and water solubility, is a promising feature for biological applications. 273,290,295 Diamond, another amorphous form of carbon, is majorly known for its remarkable hardness. Diamond exhibits insulating properties as it is composed of saturated sp 3 -hybridized carbon atoms in a tetrahedral geometry. Carbon nanodiamonds (CNDs), spherical particles possessing a diameter of about 5 nm, 290 have also gained attention because of their prosperous features. Relatively small diameters, large surface-to-volume ratio, and specific optical properties are provided. 273 Semiconductor quantum dots of CNDs are assumed to be the least toxic among all carbon allotropes discussed above. 204,296 CBMs, particularly GBMs, mostly graphene and CNTs, have been widely used in cardiac tissue engineering due to their specific properties already mentioned. Diverse mechanisms of promoting cell differentiation are the reasons for the amount of attention turned toward them, owing to their unique physical, chemical, and mechanical properties. These promoting mechanisms typically originate from features like the potency of mechanical supporting, stability in aqueous environments, the opportunity to be functionalized, large surface area, topography and presence of nanoroughnesses, and finally, yet notably, their electrical properties. 297 Herein, among all the desirable features of CBMs and different mechanisms affecting cellular interaction of CBMs, the influence of electroactivity has been reviewed in particular.

| Graphene application in cardiac repair
The first studies employing graphene nanosheets approaching cardiac tissue regeneration were conducted in the early 2010s. In a study conducted by Kim et al., 298  In another study conducted by Hitscherich and company, 305 graphene was used within a PCL substrate to prepare nanofibrous composite scaffolds for cardiac tissue engineering. As higher graphene

| Graphene oxide application in cardiac repair
As mentioned above, graphene oxide should be considered an auspicious component for the materials developed for CVDs treatment.  Contrastively, a soft injectable hydrogel utilizing π-π conjugation in which long-range electron conduction is facile was fabricated by Bao and colleagues. 155 Relative softness was achieved by emanating multi-armed polyethylene glycol diacrylate (PEGDA) from the melamine core with an π-π conjugation ring. GO incorporation aiming promoted mechanical and electrical properties were followed.
Adipose-derived stem cells (ADSCs) were then encapsulated, and an in vivo experimentation aiming for cardiac repair was conducted on Improve cell attachment, enhanced the Ca 2+ signal conduction of CM in the infarcted region, enhanced the generation of cytoskeletal structure and intercalated disc assembly hence, β-catenin signaling, were improved, which then promoted CX43 expression. Angiogenesis was also demonstrated, which was suggested to be resulting from gap junction proteins overexpression.
Moreover, highly restored cardiac function due to LV wall thickening Conductive scaffolds composed of chitosan and GO were further studied by Jiang and colleagues. 308 Electrical conductivity measurement was taken and is reported to be 1.34 Â 10 À3 S cm À1 which is likely to be favorable in cardiac tissue engineering regarding the native myocardium conductivity as already mentioned (part 4).
Later, electroactive substrates such as cardiac patches composed of polyethylene terephthalate (PET) and GO were fabricated by Ghasemi et al. 309  Notably, no necessity for special differentiated stimulating mediums is observed ( Figure 14). Silk fibroin (SF) containing rGO scaffolds was also created and evaluated by Nazari and colleagues. 321 A conductivity of 2.01 Â 10 À9 ± 3.6 Â 10 À10 Scm À1 was calculated, which was significantly higher compared to the control SF scaffolds (5.99 Â 10 À11 ± 1.2 Â 10 À11 Scm À1 ). TBX18-transfected hiPSCs were assessed for gene expression following 7 days of culture. RT-PCR evaluated C-TNT, α-MHC, and GATA-4 expression levels.

| Reduced graphene oxide application in cardiac repair
Upregulation was confirmed for each, compared to control cells.
Hence induced cardiac differentiation was achieved as a result of rGO incorporation.
Another study recently performed by Wang et al. 158

| Carbon nanotube application in cardiac repair
Compared to graphene, CNTs and CNFs were much earlier introduced to the field of cardiac regeneration. For the first time, purified singlewalled carbon nanotubes were assessed for biocompatibility with cardiac muscle cells by Garibaldi. 322 H9c2 cells were cultured in a CNTcontaining medium. Cell behavior was characterized and compared to that of untreated cells. Cell growth, survival, viability, and apoptosis were evaluated. An overall view of CNT-treated cell behavior was obtained, indicating satisfying short-term biocompatibility while longterm inconsistency was likely to result in physical rather than chemical interactions.
After the work of Garibaldi, CNTs were extensively employed as fillers for various materials applied in cardiac tissue engineering. composition and nanofibrous structure and a mean conductivity of 1.3 Â 10 À2 ± 5 Â 10 À3 S cm À1 showed excellent potential as cell proliferation and adhesion were significantly enhanced.
In another study, 328 CNT-containing electrospun scaffolds were synthesized based on chitosan and polyvinyl alcohol, and the calculated value for electrical conductivity was 3.4 Â 10 À6 S cm À1 .
A 3D printed conductive cardiac patch with an electrical conductivity of 4.3 Â 10 À1 S cm À1 was also made. 340   Furthermore, the endothelialized myocardium was constructed using this hybrid strategy via the coculture of CMs on the NFYs-NET layer and endothelial cells within the hydrogel shell. Therefore, these 3D hybrid scaffolds, containing NFYs-NET layer inducing cellular orientation, maturation, anisotropy, and hydrogel shell providing a suitable 3D environment for endothelialization, have great potential in engineering 3D cardiac anisotropy. 113

| Carbon nanofiber application in cardiac repair
Carbon nanofibers combined with different biomaterials were also earlier used for various tissue regeneration studies. 345,346 However, its entry into the cardiac area was delayed until Stout et al. 194,347 made the first use of its conductivity and cytocompatibility. Myocardial tissue repair induction potency of Poly(lactic-co-glycolic acid) (PLGA)-CNF composites were evaluated. Evaluations approved the ascending behavior of electrical conductivity as a result of increased CNF weight ratios within composites. Human CMs and rat neuroblastoma cells were cultured for in vitro cell culture assays. Results showed that cells density, as well as their proliferation rate, were both remarkably increased within composites referenced to pure PLGA. The promising results were likely to be attributed to the fabricated substrates' specific topography, roughness, and favorable electrical properties.
Later, they conducted a modified version of the previous study, utilizing a continuous electrical stimulation for 1, 3, and 5 days. 348 Cytocompatibility and viability assays were conducted on cultured human CMs, and the same overall trends were recorded. Interestingly, slightly promoted behaviors in all samples were observed as a consequence of electrical stimulations.
Further studies were conducted aiming to investigate the function and the mechanism of observations more precisely. Cardiac differentiation markers including troponin T, connexin 43, and α-SMA were seen to be highly expressed on prepared CNF-containing samples. 157 However, a descending manner of expression was observed in concentrations above some specific values.
Moreover, various cardiovascular cell types were also assessed for growth characteristics cultures on PLGA-CNF substrates. 349 Previously obtained results supported CMs through hindering effects on fibroblast, and endothelial cell growth was observed for both non or electrically stimulated groups. As indicated, this could have to do with the potential to impede the growth of fibrosis and noncontractile cells while favorably supporting CMs growth. However, the mechanism was not precisely figured out.
In another study, 350 the unique anisotropic structure of the native myocardium was regarded. Accordingly, aligned CNF-PLGA composites were fabricated. In this regard, a voltage was applied to the CNF containing the solution of PLGA before setting, thus achieving a proper orientation of nanofibers. Vertical and horizontal conductivity values of 1 Â 10 À3 and 2.5 Â 10 À5 Scm À1 were calculated, while randomly dispersed CNF samples showed the same 7.5 Â 10 À4 Scm À1 in both directions. Better adhesion and a high rate of cell proliferation were achieved on anisotropic substrates, presumably due to the specific established electrical and mechanical properties, which further improve cell-to-cell communications.